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RNA methylation m6A detection method and reagent kit

A detection kit and detection method technology, applied in the field of molecular biology, can solve the problems of inability to quantify modified sites and inaccurate positioning of adenine nucleotides, etc., achieve repeatability and reliability, save experimental funds, and be easy to operate Effect

Pending Publication Date: 2020-11-03
广州烨善生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these techniques make it possible to analyze the m6A epigenetic transcription profile, they can neither pinpoint the adenine nucleotides that actually undergo m6A modification in the MeRIP-Seq peak, nor quantify each modification site.

Method used

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  • RNA methylation m6A detection method and reagent kit
  • RNA methylation m6A detection method and reagent kit
  • RNA methylation m6A detection method and reagent kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Extract human breast cancer cell RNA from serum, take 200ng of human breast cancer cell RNA, and take the following steps for processing:

[0049] 1 Sample collection and preparation

[0050] 1.1 Take human breast cancer cells, lyse and extract RNA.

[0051] 1.2 Purity test: 1 μl RNA sample was diluted 50 times, and the OD value was measured on the BioPhotometer plus Eppendorf Nucleic Acid Protein Analyzer. The ratio of OD260 / OD280 was greater than 1.8, indicating that the prepared RNA was relatively pure and free of protein contamination;

[0052]1.3 Integrity detection of total RNA: take 1 μl of RNA sample, run 1% agarose gel electrophoresis at 80V×20min, observe the 5s rRNA, 18s rRNA and 28s rRNA bands of the total RNA with a gel imaging system, if the three bands are complete It proved that the extraction of total RNA was relatively complete.

[0053] 2. Experimental pretreatment steps

[0054] 2.1 Before the test, place the reagent and the sample to be tested at...

Embodiment 2

[0083] Grind the brain tissue and extract RNA, take 200ng of the obtained brain cell RNA, and take the following steps for processing:

[0084] 1 Sample collection and preparation

[0085] 1.1 Take human brain tissue cells, lyse and extract RNA.

[0086] 1.2 Purity test: 1 μl RNA sample was diluted 50 times, and the OD value was measured on the BioPhotometer plus Eppendorf Nucleic Acid Protein Analyzer. The ratio of OD260 / OD280 was greater than 1.8, indicating that the prepared RNA was relatively pure and free of protein contamination;

[0087] 1.3 Integrity detection of total RNA: take 1 μl of RNA sample, run 1% agarose gel electrophoresis at 80V×20min, observe the 5s rRNA, 18s rRNA and 28s rRNA bands of the total RNA with a gel imaging system, if the three bands are complete It proved that the extraction of total RNA was relatively complete.

[0088] 2. Experimental pretreatment steps

[0089] Operate with reference to the steps in Example 1.

[0090] 3 Sample addition a...

Embodiment 3

[0093] Take 200ng of yeast tRNA and take the following steps for processing:

[0094] 1 Sample collection and preparation

[0095] 1.1 Purity test: 1 μl RNA sample was diluted 50 times, and the OD value was measured on the BioPhotometer plus Eppendorf Nucleic Acid Protein Analyzer. The ratio of OD260 / OD280 was greater than 1.8, indicating that the prepared RNA was relatively pure and free of protein contamination;

[0096] 1.2 Integrity detection of total RNA: Take 1 μl of RNA sample, run 1% agarose gel electrophoresis at 80V×20min, observe the 5s rRNA, 18s rRNA and 28s rRNA bands of the total RNA with a gel imaging system, if the three bands are complete Prove that the total RNA extraction is relatively complete.

[0097] 2. Experimental pretreatment steps

[0098] Operate with reference to the steps in Example 1.

[0099] 3 Sample addition and incubation steps

[0100] Operate with reference to the steps in Example 1.

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Abstract

The invention relates to an RNA methylation m6A detection method and reagent kit. According to the detection method, a method of combining antibody capture and RNA reverse transcription is adopted, aprocess of synthesizing DNA by taking RNA as a template in the reverse transcription process is utilized, specific oligonucleotide primers are designed for a target gene m6A site region and coupled with biotin to detect the specified gene m6A level, and random primers are used for detecting the total RNAm6A level. Combination is realized by RNA fragments captured by an m6A antibody, streptavidin with an HRP label is combined with biotin for secondary signal amplification, absorbance is detected through TMB color development, and the level of the target gene m6A is calculated. The method for systematically detecting m6A incompletely depending on antibody immune enzyme-linked immunoassay is provided for the first time, has the advantages of high efficiency, sensitivity and the like, is low in detection cost, simple and convenient to operate and suitable for varied RNA samples, and solves the problems that a traditional detection method is limited in resolution, cannot perform m6A quantification and is high in RNA sample demanded quantity.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a method and kit for detecting RNA methylated m6A. Background technique [0002] N6-methyladenosine (m6A), which is a modification of adenine nucleotide catalyzed by adenylate methyltransferase, is widely distributed in different types of RNA. It is well known that RNA methylation plays an important role in RNA alternative splicing, transcription initiation and translation. The level of RNA methylation is closely related to biological processes such as metabolism, genetic development, and disease occurrence and development. [0003] Multiple methods have been developed to efficiently localize m6A across the transcriptome. The initial studies used immunoprecipitation, which utilizes highly specific m6A antibodies for next-generation sequencing. These methods are called MeRIP-seq. MeRIP has been improved and is now available for single-nucleotide resolution m6A mapping. This m...

Claims

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Application Information

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IPC IPC(8): C12Q1/6804
CPCC12Q1/6804C12Q2563/131C12Q2531/113C12Q2521/107C12Q2563/125C12Q2537/165
Inventor 林锦梅陈少锐
Owner 广州烨善生物科技有限公司
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